T cell recognition is one of the central driving forces of adaptive immunity and thus is essential to health in human beings and all higher vertebrates. In this renewal, we wish to continue our investigations of a T cell receptor structure and function, from the purely molecular level to its cellular context within an immunological synapse. While a great deal has been learned about T cell receptors over the past several decades, there are also many unresolved issues concerning how these molecules distinguish between the many peptide-MHC complexes that they need to survey and the role of CDR3 flexibility in binding and cross-reactivity. Also critical to understand are the role of coagonists, dimerization, and CD4/CD8 co-receptors, and how these might differ between mature T cells and thymocytes undergoing positive or negative selection. In this proposal, we wish to take advantage of the progress we have made during the last granting period to address these longstanding issues. Specifically, in Aim 1, we take advantage of our recent success in obtaining crystal structures of the first cytochrome c/I-Ek specific TCRs to pursue structures of these molecules in complex with their agonist- MHC ligands and lower affinity co-agonist and positive-selecting ligands. We also will investigate cross reactivity in this system and the role of CDR3 flexibility. In Aim 2, we exploit our novel FRET assay for TCR binding in situ to investigate a broad range of ligand interactions of mature T cells and thymocytes in their native context. We have also combined this FRET assay with our photocaging technique to investigate TCR engagement and signaling in a synapse on a subsecond time scale. In Aim 3, we propose to extend this in situ FRET approach to analyze CD4 and CD8 interactions with specific pMHC ligands, which promises to provide valuable information on how these molecules function. In Aim 4, we follow up on our recent demonstration that TCRs are expressed on distinct protein islands on T cell surfaces and that these TCR-containing structures join together with LAT-containing islands during T cell activation to form signaling microclusters. It is of considerable interest what other molecules involved in signaling inhabit these particular protein islands and whether some, such as ZAP-70, are able to migrate from one to another during the activation process. Taken together, these studies will build judiciously on the progress that we have made and address a number of the very important questions about T cell receptors and how they work.